The human immune system is a complex arrangement of cells and molecules that maintain immune homeostasis to preserve the integrity of the organism by elimination of all elements judged to be dangerous. Responses in the immune system may generally be divided into two arms, referred to as “innate immunity” and “adaptive immunity.”
The innate arm of the immune system is a nonspecific fast response to pathogens that are predominantly responsible for an initial inflammatory response via a number of soluble factors, including the complement system and the chemokine/cytokine system; and a number of specialized cell types, including mast cells, macrophages, dendritic cells (DCs), and natural killer cells (NKs).
The adaptive immune arm involves a specific, delayed and longer-lasting response by various types of cells that create long-term immunological memory against a specific antigen. It can be further subdivided into cellular and humoral branches, the former largely mediated by T cells and the latter by B cells. T cells further can be categorized by the expression of CD4+ molecules or the expression of CD8+ molecules, the latter of which allows for the identification of CD8+ cytotoxic T lymphocytes (CTLs).
A third arm of the immune system involves lineage members of the adaptive arm that have effector functions in the inate arm, therefore bridging the gap between the innate and adaptive immune response. These include cells such as γδ T cells and T cells with limited T cell receptor repertoires, such as natural killer T (NKT) cells and mucosal-associated invariant T (MAIT) cells. The third arm will be referred to herein as “innate-like immunity.”
The three arms of immunity do not operate independently of each other, but rather work together to elicit effective immune responses. Because the initiation of an adaptive immune response requires some time, innate immunity and innate-like immunity provide the first line of defense during the critical period just after the host's exposure to a pathogen.
Components of the Immune System
The immune system comprises cellular interactions that occur through specific receptor-ligand pairs, which signal in both directions, so that each cell receives instructions based on the temporal and spatial distribution of those signals.
Cells of the immune system include lymphocytes, monocytes/macrophages, dendritic cells, the closely related Langerhans cells, natural killer (NK) cells, mast cells, basophils, and other members of the myeloid lineage of cells. In addition, a series of specialized epithelial and stromal cells provide the anatomic environment in which immunity occurs, often by secreting critical factors that regulate growth and/or gene activation in cells of the immune system, which also play direct roles in the induction and effector phases of the response. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).
The cells of the immune system are found in peripheral organized tissues, such as the spleen, lymph nodes, Peyer's patches of the intestine and tonsils. Lymphocytes also are found in the central lymphoid organs, the thymus, and bone marrow, where they undergo developmental steps that equip them to mediate the myriad responses of the mature immune system. A substantial portion of lymphocytes and macrophages comprise a recirculating pool of cells found in the blood and lymph, providing the means to deliver immunocompetent cells to sites where they are needed and to allow immunity that is generated locally to become generalized (Id.).
Leukocytes derived from the myeloid or lymphoid lineage provide either innate or specific adaptive immunity. Myeloid cells include highly phagocytic, motile neutrophils, monocytes, and macrophages that provide a first line of defense against most pathogens. Other myeloid cells, including eosinophils, basophils, and their tissue counterparts, mast cells, are involved in defense against parasites and in the genesis of allergic reactions. Lymphocytes regulate the action of other leukocytes and generate specific immune responses that prevent chronic or recurrent infections (Id.).
The Complement System. The complement system, a part of innate immunity, comprises over 30 different proteins that circulate in blood plasma. In the absence of an infection, the complement proteins circulate in an inactive form. In the presence of a pathogen, the complement proteins become activated to kill the pathogen either directly or by facilitating phagocytosis. There are two pathways in which the complement system acts on pathogens: the classical pathway, involving antibody-dependent cell mediated cytotoxicity; and the alternative pathway, involving complement dependent cell cytotoxicity. (Ricklin, Daniel, et al. “Complement: a Key System for Immune Surveillance and Homeostasis.” Nature Immunology, U.S. National Library of Medicine, September 2010, www.ncbi.nlm.nih.gov/pmc/articles/PMC2924908/).
Antibody-dependent cell mediated cytotoxicity (ADCC) is a mechanism by which effector cells of the immune system actively lyse target cells that have been bound by antibodies. The ADCC killing mechanism of an antibody-coated target cell by a cytotoxic effector cell is through a nonphagocytic process. This process involves the release of the content of cytotoxic granules or the expression of cell death-inducing molecules. ADCC is triggered through interaction of target-bound antibodies (belonging to IgG or IgA or IgE classes) with certain Fc receptor glycoproteins present on the effector cell surface that bind the Fc region of immunoglobulins (Ig). Effector cells that mediate ADCC include natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils and dendritic cells. ADCC is dependent on a number of parameters, such as density and stability of the antigen on the surface of the target cell, antibody affinity, and FcR-binding affinity.
In contrast with ADCC, complement dependent cell cytotoxicity (CDCC) is a process of the immune system that kills pathogens by damaging target cell membrane without the involvement of antibodies. This alternative pathway is initiated by spontaneous hydrolysis and activation of the complement component C3, which binds directly to microbial surfaces. Alternatively, the lectin pathway is initiated by soluble carbohydrate binding proteins that bind to specific carbohydrate molecules on microbial surfaces.
Each of the ADCC and CDCC mechanisms generates a C3 convertase that cleaves C3, leaving behind C3b bound to the pathogen's surface and releasing C3a. This results in a number of cellular activities, including activation of the complement cascade, recruitment of phagocytic cells to the site of an infection, phagocytosis of pathogens by immune cells, and/or formation of a membrane attack complex (MAC) that disrupts pathogen cell membrane and causes cell lysis.
Immune Response
Generally speaking, immune responses are initiated by an encounter between an individual and a foreign substance, e.g., an infectious microorganism. The infected individual rapidly responds with both a humoral immune response with the production of antibody molecules specific for the antigenic determinants/epitopes of the immunogen, and a cell mediated immune response with the expansion and differentiation of antigen-specific regulatory and effector T-lymphocytes, including cells that produce cytokines and killer T cells, capable of lysing infected cells. Primary immunization with a given microorganism evokes antibodies and T cells that are specific for the antigenic determinants/epitopes found on that microorganism, but that usually fail to recognize or recognize only poorly antigenic determinants expressed by unrelated microbes (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).
As a consequence of this initial response, the immunized individual develops a state of immunologic memory. If the same or a closely related microorganism is encountered again, a secondary response ensues. This secondary response generally consists of an antibody response that is more rapid, greater in magnitude and composed of antibodies that bind to the antigen with greater affinity and that are more effective in clearing the microbe from the body, and a similarly enhanced and often more effective T-cell response. However, immune responses against infectious agents do not always lead to elimination of the pathogen (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).
Immune Homeostasis
The immune system is a tightly regulated network that is typically able to maintain homeostasis under normal physiological conditions in that the various actors of the immune system act cooperatively to avoid immune disequilibrium. Normally, when challenged with a foreign antigen, specific appropriate responses are initiated that are aimed at restoring equilibrium. However, under certain circumstances, this balance is not maintained and immune responses either under- or over-react. Cancer is an example of a situation where the immune response can be inefficient or unresponsive, resulting in uncontrolled growth of the cancer cells. Conversely, when the immune response over-reacts, this can result in conditions such as autoimmunity, chronic inflammation, and/or pathology following infection.
Immune Tolerance
The immune system is tolerant of self-antigens, i.e., it can discriminate between antigenic determinants expressed on foreign substances, and antigenic determinants expressed by tissues of the host. The capacity of the system to ignore host antigens, referred to as immune tolerance or immunological tolerance, is an active process involving the elimination or inactivation of cells that could recognize self-antigens through immunologic tolerance (Fundamental immunology, 4th Edn, William E. Paul, Ed. Lippincott-Raven Publishers, Philadelphia, (1999), at p. 2).
Innate immune cells recognize and discriminate between self and non-self through three distinct mechanisms: 1) innate leukocytes can recognize “nonself” from “non-infectious self” by recognizing conserved products not expressed by the host; 2) innate immune cells can recognize “missing self” by recognizing self-proteins that are specific to the host and absent from pathogens; 3) innate immune cells can also recognize “altered self” by recognizing abnormal cell markers that are upregulated due to infection or cellular transformation. (Spear, Paul, et al. “NKG2D Ligands as Therapeutic Targets.” Cancer Immunity, Academy of Cancer Immunology, 1 May 2013, www.ncbi.nlm.nih.gov/pmc/articles/PMC3700746/).
Immune tolerance is classified into 1) central tolerance or 2) peripheral tolerance, depending on where the state is originally induced, i.e., whether it is in the thymus and bone marrow (central) or in other tissues and lymph nodes (peripheral). The biological mechanisms by which these forms of tolerance are established are distinct, but the resulting effect is similar (Raker V. K. et al. Front Immunol, Vol., 6(569): 1-11, (2015)).
Central tolerance, the principal way in which the immune system is educated to discriminate self-molecules from non-self-molecules, is established by deleting autoreactive lymphocyte clones at a point before they mature into fully immunocompetent cells. It occurs during lymphocyte development in the thymus and bone marrow for T and B lymphocytes, respectively (Sprent J. et al. Philos Trans R Soc Lond B Biol Sci, Vol. 356(1409): 609-616, (2001)). In these tissues, maturing lymphocytes are exposed to self-antigens presented by thymic epithelial cells and thymic dendritic cells, or bone marrow cells. Self-antigens are present due to endogenous expression, importation of antigen from peripheral sites via circulating blood, and in the case of thymic stromal cells, expression of proteins of other non-thymic tissues by the action of the transcription factor AIRE (Murphy, Kenneth. Janeway's Immunobiology: 8th ed. Chapter 15: Garland Science. (2012), pp. 611-668; see also, Klein L. Cell, Vol. 163(4):794-795, (2015)). Those lymphocytes that have receptors that bind strongly to self-antigens are removed by means of apoptosis of the autoreactive cells, or by induction of anergy (Id. at pp. 275-334). Weakly autoreactive B cells may also remain in a state of immunological inactivity where they do not respond to stimulation of their B cell receptor. Some weakly self-recognizing T cells are alternatively differentiated into natural regulatory T cells (nTreg cells), which act as sentinels in the periphery to lower potential instances of T cell autoreactivity (Id. at pp. 611-668).
The deletion threshold is more stringent for T cells than for B cells, since T cells are the main populations of cells that can cause direct tissue damage. Furthermore, it is more advantageous for the organism to let its B cells recognize a wider variety of antigens so that they can elicit antibodies against a greater diversity of pathogens. Since B cells can only be fully activated after confirmation by more self-restricted T cells that recognize the same antigen, autoreactivity is held in great check (Id. at pp. 275-334).
This process of negative selection ensures that T and B cells that potentially may initiate a potent immune response to the individual's own tissues are destroyed while preserving the ability to recognize foreign antigens. Lymphocyte development and education is most active in fetal development, but continues throughout life as immature lymphocytes are generated, slowing as the thymus degenerates and the bone marrow shrinks in the adult life (Id. at pp. 275-334; see also Jiang T. T. J Immunol., Vol. 192(11): 4949-4956, (2014)).
Peripheral tolerance develops after T and B cells mature and enter the peripheral tissues and lymph nodes (Murphy, Kenneth. Janeway's Immunobiology: 8th ed. Chapter 8: Garland Sciences. pp. 275-334). It is set forth by a number of overlapping mechanisms that predominantly involve control at the level of T cells, especially CD4+ helper T cells, which orchestrate immune responses and give B cells the confirmatory signals that the B cells need in order to progress to produce antibodies. Inappropriate reactivity toward a normal self-antigen that was not eliminated in the thymus can occur, since the T cells that leave the thymus are relatively, but not completely, safe. Some will have TCRs that can respond to self-antigens that the T cell did not encounter in the thymus (Id.). Those self-reactive T cells that escape intra-thymic negative selection in the thymus can inflict cell injury unless they are deleted in the peripheral tissue chiefly by nTreg cells.
Autoimmune regulator (Aire), usually expressed in thymic medullary epithelial cells, plays a role in immune tolerance by mediating ectopic expression of peripheral self-antigens and mediating the deletion of auto-reactive T cells (Metzger T. C., et al. Immunol. Rev. 2011, 241: 89-103, (2011)).
Appropriate reactivity towards certain antigens can also be suppressed by induction of tolerance after repeated exposure. Naïve CD4+ helper T cells differentiate into induced Treg cells (iTreg cells) in the peripheral tissue, or accordingly, in nearby lymphoid tissue (lymph nodes, mucosal-associated lymphoid tissue, etc.). This differentiation is mediated by IL-2 produced upon T cell-activation, and TGF-β from any of a variety of sources, including tolerizing dendritic cells (DCs) or other antigen presenting cells (Curotto de Lafaille et al. Immunity, 30(6): 626-635, (2009)).
Immunity and Cancer
Immune Tolerance of Cancer
Cancer is characterized by genetic instability of particular cells, but has also been described as a disorder of the immune system, based on the fact that the immune system fails, at least in certain segments of the afflicted human population, to respond optimally to cancerous cells that have taken on a distinctly non-self phenotype that should be recognized as foreign. Several reasons have been advanced to explain the basis of this observation. For example, first, cancer cells consist mainly of self-antigens, in striking contrast to the situation with infectious organisms. Some antigens that are classified as cancer antigens are actually normal antigens that are overexpressed, or normal antigens that have a mutation in only one or two amino acids in the polypeptide chain. Second, cancer cells down-regulate MHCs, and thus do not much present tumor cell-derived peptides by way of MHC. Third, cancer cells, and associated tumor-associated macrophages, express cytokines that dampen the immune response (see, e.g., Yu et al (2007) Nature Rev. Immunol. 7:41-51). This dampening is caused, for example, by the secretion of interleukin-10 (IL-10) by the cancer cells or by the associated macrophages. Fourth, unlike the situation with infections, cancer cells do not provide any immune adjuvant. Pathogens express a variety of naturally-occurring immune adjuvants, which take the form of TLR agonists and NOD agonists (see, e.g., Kleinnijenhuis et al (2011) Clin. Dev. Immunol. 405310 (12 pages)). Generally, optimal activation of dendritic cells requires contact of an immune adjuvant with one or more TLRs expressed by the dendritic cell. Without activation of the dendritic cell, contact between the dendritic cell and T cells (immune synapse) fails to result in optimal activation of the T cell.
Tumor Immune Surveillance and Immune Editing
While a functional cancer immunosurveillance process indeed exists that acts as an extrinsic tumor suppressor, it has become clear that the immune system can facilitate tumor progression, at least in part, by sculpting the immunogenic phenotype of tumors as they develop. This so-called “tumor immune editing” is divided into three phases: an elimination phase, an equilibrium phase, and an escape phase. The elimination phase, also known as immune surveillance, is the process by which the immune system identifies cancerous or pre-cancerous cells and eliminates them before they grow out of control. This phase can be complete when all cancerous or precancerous cells are eliminated. If some tumor cells are not eliminated, a temporary state of equilibrium may be achieved between the immune system and tumor cell growth. In this equilibrium phase, tumors cells can either remain dormant or continue to evolve by accumulating further changes to genomic DNA that can modulate the antigens they present. During this process, the immune system exerts a selective pressure on evolving cells, whereby the tumor cells that are less able to be recognized have a survival advantage. Eventually the immune response is unable to recognize cells of the tumor, resulting in the transition to the escape phase, where tumor cells progressively grow out of control. (Dunn, G P et al., Ann. Rev. Immunol. (2004): 329-60).
Tumor Immunology
Tumors are able to progress and evolve by numerous evasion mechanisms.
For example, tumors are able to evolve under selective pressure from the immune response to selectively lose receptors that activate anti-tumor immune cells. For example, it has been reported that tumors that are NKG2D ligand-deficient in mice that are NKG2D expressing have been able to persist despite the loss of other tumor cells. (Marcus, Assaf, et al. “Recognition of Tumors by the Innate Immune System and Natural Killer Cells.” Advances in Immunology, U.S. National Library of Medicine, 2014, www.ncbi.nlm.nih.gov/pmc/articles/PMC4228931/).
Tumors also shed ligands that activate anti-tumor immune cells through a variety of techniques, such as alternative splicing, cleavage, proteolytic shedding, or exosome secretion. This can be seen in the increase of soluble ligands, such as MIC (MHC class I-related molecules distantly related to the MHC class I proteins) and UL16-binding proteins (ULBPs) which bind to MICB), that have been identified in the sera of patients with various tumor types, including breast, lung, colon, and obarious carinomal, glioma, neuroblastoma, leukemia, and melanoma. The shedding of ligands and the existence of soluable ligands in the surrounding reaction environment can result in several distinct effects. First, it decreases the level of activating ligands on the cell surface and thus reducing tumor cell susceptibility to attack by lymphocytes. For example, it has been postulated that the shedding of NKG2D ligands from tumor cells reduces their ability to be cytolytic attacked by NKs or T cells. Alternatively, the existence of soluable ligands in the reaction environment may desensitize NKs by binding to ligand receptors on lymphocytes and preventing interactions necessary to induce cytotoxic activity on tumor cells. Id. Soluable ligands are also thought to downregulate the expression of their receptors. For example, cancer patients with elevated soluble MICA in their serum exhibited strongly reduced NKG2D staning of their peripheral blood CD8+ T cells. Id. Soluble ligands along with exosomes have also been postulated to bundle together and act in concert to impact lymphocyte immune responses. Id.
Similarly, tumors can lose the ability to express receptors and/or shed them in an effort to evade cell death. For example, tumors can evade immune recognition through disrupting MHC class I restricted antigen processing through the loss of class I itself or components in the class I pathway. Some melanomas have lost cell surface expression of MHC class I through defective expression of β2 microglobulin (β2M), which is required for stable assembly of class I, or defective expression of the transporter associated with tumor antigen processing (TAP). (Alberts, D. S., and L. M. Hess, editors. FUNDAMENTALS OF CANCER PREVENTION. SPRINGER NATURE, 2019. Pps. 79-108).
Tumor Microenvironment
The tumor microenvironment provides a consistently effective barrier to immune cell function, because tumors actively downregulate all phases of anti-tumor immune responses using a spectrum of different strategies and mechanisms. Many molecular mechanisms that cause dysfunction of immune cells in the tumor microenvironment have been identified, including those directly mediated by factors produced by tumors, and others resulting from alterations of normal tissue homeostasis in the presence of cancer. Most human tumors appear to be able to interfere with one or more stages of immune cell development, differentiation, migration, cytotoxicity and other effector functions (T L Whiteside, The tumor microenvironment and its role in promoting tumor growth, Oncogene (2008) 27, 5904-5912).
One such mechanism involves accumulation in tumors of regulatory T cells (Tregs) (CD4+CD25bright Foxp3+) and myeloid-derived cells (CD34+CD33+CD13+CD11b+CD15−), which are common features of human tumors, and have been linked to poor prognosis in patients with cancer (Id.). Under normal conditions, Treg cells are involved in preventing autoimmunity, but in cancer, they expand, migrate to tumors, downregulate autologous effector T-cell proliferation, and suppress anti-tumor responses of both CD4+CD25− and CD8+CD25− T cells using distinct molecular pathways. The Treg cells in the tumor are a heterogeneous population of regulatory CD3+CD4+ T cells, comprising natural Treg, antigen-specific Tr1 cells, and other less well defined subsets of suppressor cells. T regulatory type 1 (Tr1) cells are induced in the tumor microenvironment, which is rich in IL-10, TGF-β, and prostaglandin E2 (PGE2), all of which have been shown to promote Tr1 generation (Id.).
Myeloid-derived suppressor cells (MDSC's), which are closely related to neutrophils and monocytes, are not present at steady state in healthy individuals, and appear in cancer and pathological conditions associated with chronic inflammation or stress. (Gabrilovich, D I., “Myeloid-derived suppressor cells,” Cancer Immunol. Res. (2017) 5(1): 3-8). They are a relatively stable, distinct state of functional activity of neutrophils and monocytes. The main functional characteristic of these cells is their potent ability to suppress various types of immune responses. MDSC consist of two large groups of cells termed granulocytic or polymorphonuclear (PMN-MDSC), which phenotypically and morphologically are similar to neutrophils; and monocytic (M-MDSC), which are phenotypically and morphologically similar to monocytes. Therefore phenotypic criteria alone are not sufficient to identify cells as MDSCs. In most types of cancer, PMN-MDSC represent more than 80% of all MDSC. In addition to these two main populations, MDSCs include a small group (less than 3%) of cells with myeloid colony forming activity representing a mixture of myeloid progenitors and precursors. Among peripheral blood mononuclear cells (PBMCs), PMN-MDSCs are defined as CD11b+CD14−CD15+ or CD11b+CD14−CD66b+, and M-MDSC as CD11b+CD14+HLA-DR−/loCD15−. Lin− (including CD3, CD14, CD15, CD19, CD56) HLA-DR−CD33+ cells contain mixed groups of MDSC comprising more immature progenitors. The term “early-stage MDSC” (e-MDSC) has been proposed for this latter population.
Although MDSCs were implicated in suppression of different cells of the immune system, the main targets of MDSCs are T cells. The main factors implicated in MDSC-mediated immune suppression include arginase (ARG1), iNOS, TGFβ, IL-10, COX2, indoleamine 2,3-dioxygenase (IDO) sequestration of cysteine, decrease of L-selectin expression by T-cells and many others. M-MDSC and PMN-MDSC utilize different mechanisms of immune suppression. M-MDSC suppress T-cell responses both in antigen-specific and non-specific manners utilizing mechanisms associated with production of NO and cytokines (reviewed in (Id., citing Gabrilovich, D E et al, Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol. (2012)12:253-68). PMN-MDSCs, on the other hand, are capable of suppressing immune responses primarily in an antigen-specific manner. Induction of antigen-specific T-cells tolerance is one of the major characteristics of these cells (Id., citing Koehn B H, et al. GVHD-associated, inflammasome-mediated loss of function in adoptively transferred myeloid-derived suppressor cells. Blood (2015) 126:1621-8; Nagaraj S, Gupta K, Pisarev V, Kinarsky L, Sherman S, Kang L, et al. Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer. Nat Med. (2007) 13: 828-35). Reactive oxygen species (ROS) production is essential for this ability. Reaction of NO with superoxide generates peroxynitrite (PNT), which directly inhibits T-cells by nitrating T-cell receptors and reducing their responsiveness to cognate antigen-MHC complexes (Id., citing Nagaraj S, et al. Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer. Nat Med. (2007) 13: 828-35). PNT also reduces the binding of antigenic peptides to MHC molecules on tumor cells (Id., citing Lu, T. et al., Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice. J. Clinical Investigation. (2011) 121: 4015-29) and blocks T-cell migration by nitrating T-cell specific chemokines (Id., citing Molon, B. et al., Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells. J Exp Med. (2011) 208: 1949-62). Besides immune suppressive mechanisms, MDSCs promote tumor progression by affecting the remodeling of the tumor microenvironment and tumor angiogenesis via production of VEGF, bFGF, Bv8, and MMP9 (Id., citing Tartour, E. et al., Angiogenesis and immunity: a bidirectional link potentially relevant for the monitoring of antiangiogenic therapy and the development of novel therapeutic combination with immunotherapy. Cancer Metastasis Rev. (2011) 30: 83-95; Casella, I., et al., Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through FM receptor. Blood. (2003) 101:1316-23; Shojaei, F. et al., G-CSF-initiated myeloid cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy in mouse models. Proc Natl Acad Sci USA. (2009) 106: 6742-7).
Mostly, dendritic cells found in the TME are immature and tunable to activate specific T cells. NKTs secrete IL-4 and IFN-γ and further upregulate CD40L, thereby inducing the maturation of DCs. DC maturation leads to increased costimulatory capacity through upregulation of CD80 and CD86, of MHC molecules, and by producing the pro-inflammatory cytokine IL-12 and the chemokine, CCL17. The presence of the chemokine attracts CCR4+ cells including CD8+ T cells, which then can be activated by the “licensed” DC cell. (Gottschalk et al. (2015) “The Role of Invariant Natural Killer T Cells in Dendritic Cell Licensing, Cross-Priming, and Memory CD8+ T Cell Generation.” Front Immunol 6:379).
Typically, there are two types of tumor cells in a human tumor mass: one is HLA Class I-positive and the other is HLA Class I-negative. Effective tumor immunity requires that both types of tumor cells are eliminated at once. NKTs are the only cell type that is able both to interact with immature DCs, inducing their maturation, and to augment the function of both NK and CD8+ T cells. NKTs induce maturation of DCs, allowing DCs to present tumor antigens to CD8+ T cells. The activated CD8+ T cells can then eliminate HLA Class I—positive tumor cells. NKTs also produce IFNγ which activates NKs thereby killing HLA Class I—negative tumor targets. (Terabe, M., & Berzofsky, J. A. (2012). Natural killer T cells balancing the regulation of tumor immunity. New York, N.Y.: Springer).
NKTs can form bidirectional interactions with B cells, which can present lipid antigens to some NKTs through CD1d. In return, NKTs can license B cells to effectively prime and activate antitumor CTL responses and provide B call help to enhance and sustain a humoral response. (Nair and Dhodapkar (2017). “Natural Killer T Cells in Cancer Immunotherapy.” Frontiers in Immunology 8:1178).
Tumor associated macrophages (TAMs) are prominent immunosuppressive immune cells present in the tumor microenvironment. TAMs contribute to tumor progression by enhancing angiogenesis, tumor cell invasion, suppression of NKs and T cell responses. Some NKTs have been found to o-localize with CD1d-expressing TAMs in neuroblastomas and kill TAMs in an IL-15 and CD1d-restricted manner. (Id.).
NKTs can also alter the effects of CD1d+ myeloid-derived suppressor cell (MDSC)-mediated immune suppression. MDSCs often accumulate during tumor growth and contribute to immune escape and tumor progression. Research has found that NKTs may inhibit the arginate 1 and nitrous oxide synthase-mediated suppressive activity of MDSCs. This ability to inhibit the immunosuppressive activity of MDSCs has been reported to be dependent on CD1d and CD40 interactions. (Id.).
While some NKTs can promote strong antitumor immunity, other types have been known to suppress antitumor immune responses and play more of a regulatory role, similar to Tregs and MDSCs. The balance between immunomodulating and immunosuppressive NKTs can determine whether immune responses to tumors will be activated resulting in tumor elimination, or will be suppressed, allowing the tumor to grow. (Terabe, M., & Berzofsky, J. A. (2012). Natural killer T cells balancing the regulation of tumor immunity. New York, N.Y.: Springer).
Some NKT types have been shown to promote the accumulation of MDSCs in tumor-bearing mice. NKTs have also been shown to inhibit the proinflammatory functions of other NKT cell types, conventional T cells, and DCs. One attribute of immunosuppressive NKTs is their elevated production of IL-13 and IL-4 cytokines, which are capable of skewing the cytokine response predominantly towards the tumor promoting Th2 type. Studies have shown that immunosuppressive type NKTs have been shown to suppress cytotoxic T cells through IL-13 production via an IL4R and STAT6 axis, and also induce MDSCs producing immunosuppressive cytokine TGF-B. (Nair and Dhodapkar (2017). “Natural Killer T Cells in Cancer Immunotherapy.” Frontiers in Immunology 8:1178).
It has been hypothesized that immunosuppressive NKTs when stimulated with CpG secrete IFNy instead of IL-13, therefore enhancing the activation and function of CD8+ cells and contributing to an anti-tumor effect. (Id.) Therefore, while the balance of immunosuppressive NKTs and immunomodulating NKTs is important in enhancing anti-tumor activity, another factor is the activating ligand of the NKTs itself.
Tumor Immunotherapy
Traditional chemotherapy works by killing cells that multiply quickly whether normal or cancerous. Targeted therapy works by stopping or slowing the growth or spread of cancer on a cellular level by targeting the cancer's specific genes, proteins, or the tissue environment that contributes to cancer growth and survival.
Monoclonal antibodies, for example, block a specific target on the outside of cancer cells and/or in the area around the cancer. Antibody therapies such as Trastuzumab (Herceptin®), which is effective against tumors that overexpress the HER2/neu protein, and Cetuximab (Erbitux®), an epidermal growth factor receptor inhibitor antineoplastic agent, have yielded considerable improvement in clinical outcome, as measured by, e.g. the recurrence rate, progression free survival and overall survival.
Small molecule drugs have been designed against specific targets. For example, angiogenesis inhibitors keep tissue around the tumor from making blood vessels, thereby starving the tumor (e.g., bevacizumam (Avastin®); imatinib mesylate (GLEEVEC™); tamoxifen attenuates VEGF-mediated angiogenesis (antiangiogenic effect mediated by EGF (McNamara, D A et al., Eur. J. Surg. Oncol. (2001) 27(8): 714-718)
Immunotherapy is a type of therapy that uses substances to stimulate or suppress the immune system to help the body fight cancer, infection and other diseases. Some types of immunotherapy only target certain cells of the immune system. Others affect the immune system more generally.
Anti-cancer immunotherapy has been an unattained goal for many years. One difficulty is that target antigens are often tissue specific molecules found on both cancer cells and normal cells, and either do not elicit immunity or show non-specificity regarding cell killing (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). Furthermore, tumor cells have features that make immune recognition difficult, such as loss of expression of antigens that elicit immune response, lack of major histocompatibility (MHC) class II, and downregulation of MHC class I expression. These features can lead to non-recognition of tumor cells by both CD4+ and CD8+ T cells (Id.). Tumors may also evade detection through active mechanisms, such as the production of immunosuppressive cytokines (Id.)).
Dendritic cell vaccines are vaccines made of antigens and dendritic antigen-presenting cells (APCs). Vaccination strategies involving DCs to induce tumor-specific effector T cells that can reduce the tumor mass specifically and that can induce immunological memory to control tumor relapse have been developed. For example, DCs generated ex vivo by culturing hematopoietic progenitor cells or monocytes with cytokine combinations have been tested as therapeutic vaccines in cancer patients for more than a decade (Ueno H, et al., Immunol. Rev. (2010) 234: 199-212). Treatment of metastatic prostate cancer with sipuleucel-T (also known as APC 8015), a cellular product based on enriched blood APCs that are briefly cultured with a fusion protein of prostatic acid phosphatase (PAP) and granulocyte macrophage colony-stimulating factor (GM-CSF), resulted in an approximately 4-month-prolonged median survival in Phase III trials (Higano C S, et al., Cancer (2009) 115: 3670-3679; Kantoff P W, et al., N. Engl. J. Med. (2010) 363: 411-422). This study concluded that DC-based vaccines are safe and can induce the expansion of circulating CD4+ T-cells and CD8+ T-cells specific for tumor antigens. As a result of this and similar studies, sipuleucel-T has been approved by the US Food and Drug Administration (FDA) for the treatment of metastatic prostate cancer, thereby paving the clinical development and regulatory path for the next generation of cellular immunotherapy products (Palucka K and Banchereau J, Nature Reviews Cancer (April 2012) 12: 265-276).
DC-tumor cell fusions have been developed to generate hybrid cells that express the relevant tumor associated antigens derived from the parent tumor cells, and that also have the ability to process and present such antigens to appropriate cells of the immune system. Such DC-tumor cell fusions provide a greater variety of tumor antigens, but have met with limited success in human trials, likely due to the autologous components required, the heterogeneity of the product caused by maturation of DC cells, and variations in antigen loading (Browning, M., Antigen presenting cell/tumor cell fusion vaccines for cancer, Human Vaccines & Immunotherapeutics 9:7, 1545-1548; July 2013; Butterfield, L., Dendritic Cells in Cancer Immunotherapy Clinical Trials: Are We Making Progress?, Frontiers of Immunology, 2013 4: 454).
Immune checkpoint inhibitors (e.g., PD-1 and CTLA4 inhibitors) have been reported to block discrete checkpoints in an active host immune response allowing an endogenous anti-cancer immune response to be sustained. As used herein, the term “immune checkpoints” refers to the array of inhibitory pathways necessary for maintaining self-tolerance and that modulate the duration and extent of immune responses to minimize damage to normal tissue. Immune checkpoint molecules such as PD-1, PD-L1, CTLA-4 are cell surface signaling receptors that play a role in modulating the T-cell response in the tumor microenvironment. Tumor cells have been shown to utilize these checkpoints to their benefit by up-regulating their expression and activity. With the tumor cell's ability to commandeer some immune checkpoint pathways as a mechanism of immune resistance, it has been hypothesized that checkpoint inhibitors that bind to molecules of immune cells to activate or inactivate them may relieve the inhibition of an immune response. Recent discoveries have identified immune checkpoints or targets, like PD-1, PD-L1, PD-L2, CTLA4, TIGIT, TIM-3, LAG-3, CCR4, OX40, OX40L, IDO, and A2AR, as proteins responsible for immune evasion. Specific immune checkpoint inhibitors, including antibodies against CTLA-4, PD-1 receptor or its ligand PD-L1 have produced impressive results in the clinic in a range of cancers, leading to FDA approvals for YERVOY™ (Ipilimumab; CTLA-4 antagonist), OPDIVO™ (Nivolumab; PD-1 antagonist) and KEYTRUDA™ (Pembrolizumab; PD-1 antagonist) in multiple tumor indications and with ongoing registration trials in many more.
For example, TIGIT, a member of the Ig super family and an immune inhibitory receptor, is overexpressed on tumor antigen-specific CD8+ T cells and CD8+ TILs and plays a key role in the suppression of T-cell proliferation and activation; it is involved in tumor cell immune evasion, and the inhibition of antiviral immune responses. Anti-TIGIT monoclonal antibody OMP-313M32 targets this immune checkpoint and prevents T cell downregulation. Upon administration, anti-TIGIT monoclonal antibody OMP-313M32 binds to TIGIT expressed on various immune cells, including T cells, and prevents the interaction of TIGIT with its ligands CD112 (nectin-2; poliovirus receptor related-2; PVRL2) and CD155 (poliovirus receptor; PVR; nectin-like protein 5; NECL-5). This leaves CD112 and CD155 free to interact with the costimulatory receptor CD226 (DNAX Accessory molecule-1; DNAM-1), which is expressed on immune cells, such as natural killer (NK) cells and CD8-positive T cells, and leads to CD226 dimerization and CD226-mediated signaling. This activates the immune system to exert a T-cell-mediated immune response against cancer cells.
TIM-3, a transmembrane protein and immune checkpoint receptor, is associated with tumor-mediated immune suppression. Anti-TIM-3 monoclonal antibody TSR-022, a monoclonal antibody against the inhibitory T-cell receptor, T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3; TIM3; hepatitis A virus cellular receptor 2; HAVCR2), and anti-TIM-3 antibody BMS-986258, an antibody against TIM-3, have potential immune checkpoint inhibitory and antineoplastic activities. Upon administration, the anti-TIM-3 monoclonal antibody TSR-022 binds to TIM-3 expressed on certain T cells, including tumor infiltrating lymphocytes (TILs). This abrogates T-cell inhibition, activates antigen-specific T lymphocytes and enhances cytotoxic T-cell-mediated tumor cell lysis, which results in a reduction in tumor growth.
LAG-3 is a member of the immunoglobulin superfamily (IgSF) and binds to major histocompatibility complex (MHC) class II. LAG-3 expression on TILs is associated with tumor-mediated immune suppression.
Relatlimab (previously known as BMS-986016, Bristol-Myers Squibb) is a monoclonal antibody directed against the inhibitor receptor lymphocyte activation gene-3 (LAG-3), with potential immune checkpoint inhibitory and antineoplastic activities. Upon administration, relatlimab binds to LAG-3 on tumor infiltrating lymphocytes (TILs), which may activate antigen-specific T lymphocytes and enhance cytotoxic T cell-mediated tumor cell lysis, which leads to a reduction in tumor growth.
Anti-LAG-3 monoclonal antibody LAG525 is a humanized monoclonal antibody directed against the inhibitory receptor lymphocyte activation gene-3 (LAG-3), with potential immune checkpoint inhibitory and antineoplastic activities. Upon administration, the anti-LAG-3 monoclonal antibody LAG525 binds to LAG-3 expressed on tumor-infiltrating lymphocytes (TILs) and blocks its binding with major histocompatibility complex (MHC) class II molecules expressed on tumor cells. This activates antigen-specific T-lymphocytes and enhances cytotoxic T-cell-mediated tumor cell lysis, which leads to a reduction in tumor growth. LAG-3, a member of the immunoglobulin superfamily (IgSF) and expressed on various immune cells, negatively regulates cellular proliferation and activation of T-cells. Its expression on TILs is associated with tumor-mediated immune suppression.
Anti-LAG3 monoclonal antibody TSR-033 is a humanized, immunoglobulin G4 (IgG4) monoclonal antibody directed against the inhibitory receptor lymphocyte activation gene 3 protein (LAG3; LAG-3), with potential immune checkpoint inhibitory and antineoplastic activities.
TIGIT targeting agent MK-7684 is an antagonistic agent targeting the co-inhibitory molecule and immune checkpoint inhibitor T-cell immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT; T-cell immunoreceptor with Ig and ITIM domains; T-cell immunoglobulin and ITIM domain), with potential immune checkpoint inhibitory and antineoplastic activities. Upon administration, MK-7684 targets and binds to TIGIT expressed on various immune cells, particularly on tumor-infiltrating T lymphocytes (TILs) and natural killer (NK) cells, thereby preventing the interaction of TIGIT with its ligands CD112 (nectin-2; poliovirus receptor related-2; PVRL2) and CD155 (poliovirus receptor; PVR; nectin-like protein 5; NECL-5), which are expressed on T cells, NK cells and certain cancer cells. This enhances the interaction of CD112 and CD155 with the costimulatory receptor CD226 (DNAX Accessory molecule-1; DNAM-1), which is expressed on immune cells, such as NK cells and CD8+ T cells, and activates CD226-mediated signaling. This activates the immune system to exert a T-cell-mediated immune response against cancer cells.
This method of therapy, however, can only be successful if a pre-existing antitumor immune response is present within a patient (Pardoll, D., The blockade of immune checkpoints in cancer immunotherapy, Nature Reviews: Cancer, Vol. 12, April 2012, 253).
Chimeric antigen receptor T-cell therapy (CAR-T), attempts to use synthetic biology to redirect T-cells to specific cell surface tumor antigens. Genetic modification of T-cells is used to confer tumor antigen recognition by transgenic expression of a chimeric antigen receptor (CAR). CARs are engineered molecules that can be introduced into T cells to enable them to target tumor antigens (Frey, N. V., Porter, D. L., The Promise of Chimeric Antigen Receptor T-Cell Therapy, Oncology (2016); 30(1)) pii 219281). CAR T cells have been shown to have some efficacy against hematologic malignancies and to a lesser extent solid tumors. CAR T therapy, however, has been shown to cause several types of toxicities, including cytokine release syndrome, neurological toxicity, non-tumor recognition, and anaphylaxis (Bonifant C L, et al., Toxicity and management in CAR T-cell therapy, Molecular Therapy—Oncolytics (2016) 3, 16011).
Cellular vaccines have also been proposed as a cancer treatment. GVAX™ is a GM-CSF gene transduced tumor vaccine within either an autologous or allogeneic population of tumor cells. It was believed that GM-CSF secretion of genetically modified tumor cells would stimulate cytokine release at the vaccine site to activate antigen presenting cells to induce a tumor specific cellular immune response (Eager, R. & Nemunaitis, J., GM-CSF Gene-Transduced Tumor Vaccines, Molecular Therapy, Vol. 12, No. 1, 18 (July 2005)). However, GVAX™ yielded only limited clinical responses.
Tumor cell lines possess a broad array of antigens, many of which are common to a particular tumor type, as well as some that are shared across tumors. Many immunomodulatory components defined as a result of decades of research can be used to genetically engineer these tumor cell lines. An allogeneic approach to immunoactivation in the context of such allogeneic tumor cell lines modified to express at least 2/3/4 immunomodulators has been described.
The described invention provides a method for effective tumor cell killing through adoptive transfer of in vitro (or in vivo) activated mononuclear cells. The method described herein involves the in vitro immune activation of mononuclear cells following their co-incubation with allogeneic engineered leukocyte stimulator cells (ENLST™ cells) encoding at least three (3) immunomodulator peptides. Through cell contact, the mononuclear cells are stimulated to differentiation, proliferate and acquire an activated phenotype. The activated mononuclear cells, or subpopulations thereof comprised of serial killer cells are useful for passive adoptive transfer of the cell product to the patient. Since the cells are activated in a physiologic manner, the stimulated cells retain homeostatic control mechanisms of their cell type. Optionally, immortalizing the subpopulations comprising serial killer cells represents the possibility of creating an infinite supply.